Method of treatment of type-1 diabetes with humanin analogues

ABSTRACT

The present invention is based on the discovery that Humanin and humanin analogues protect pancreatic beta cells in vitro and in vivo from apoptosis. Accordingly, humanin and its analogues are useful for preventing and treating diabetes and promoting beta cell survival in a number of applications.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/210,856 filed on Sep. 15, 2008, now U.S. Pat. No. 7,998,928issued Aug. 16, 2011 and claims priority to U.S. Provisional ApplicationNo. 60/972,596 filed on Sep. 14, 2007, all of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Type I diabetes is an autoimmune disease characterized by theprogressive destruction of pancreatic beta cells following infiltrationof the islet by lymphocytes. This results in insulin deficiency.

Apoptosis is the primary mode of beta cell death during development ofType-1 diabetes (O'Brien et al. (1997) Diabetes 46:750-57). IL-1,TNF-alpha and IFN-gamma are released by T cells and macrophages duringthis autoimmune response and are important mediators of beta celldestruction (Eizirik and Mandrup-Poulsen (2001) Diabetologia44:2115-2133).

Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) induces apoptosisin a manner unrelated to its IGF binding (Rajah et al. (1997) J BiolChem. 272:12181-88). Pro-inflammatory Th1 cytokines increasesintranuclear aggregation of endogenous IGFBP-3 and addition of exogenousIGFBP-3 to beta cells induces apoptosis (Shim et al. (2004) Growth HormIGF Res. 14:216-25). IGFBP-3 is one of a number of peptides thatincludes insulin, leptin, adiponectin, and resistin, that have beenshown to act in the central nervous system to regulate glucosemetabolism (Muse et al. (20070 J Clin Invest. 117:1670-78; Obici et al.(2002) Nat Med 8:1376-82). Beta cell loss by apoptosis also occurs afterislet graft (Paraskevas et al. (1999) FEBS Lett. 455:203-8); Tobiasch etal. (2001) J Investig Med. 49:566-71). Recent studies have demonstratedthat isolated human islets express the pro-apoptotic protein Bax athigher level than the anti-apoptotic protein Bcl-2 (Thomas et al. (2002)Transplantation 74:1489).

Over a million people in the U.S. have type I diabetes. According to theAmerican Diabetes Association, the disease causes thousands of deathsevery year and costs more than $20 billion annually. There is currentlyno effective therapeutic or preventative agent available for Type Idiabetes.

Humanin (HN) is a recently described 24-amino acid peptide. HN wasindependently cloned as a neuroprotective protein, a BAX antagonist, andas an IGFBP-3 binding partner in a yeast two-hybrid assay (Hashimoto etal. (2001) Proc. Natl. Acad. Sci. USA 98:6336-41; Guo et al. (2003)Nature 423:456-61; Ikonen et al. (2003) Proc. Natl. Acad. Sci USA100:13042-47). HN is transcribed from an open reading frame within themitochondrial 16S ribosomal RNA in mammals (Hashimoto et al., 2001). HNis both an intracellular and secreted protein. HN has been detected innormal mouse testis and colon (by immunoblot and immunohistochemicalanalyses), as well as in cerebrospinal fluid (CSF), seminal fluid, andserum. Levels in CSF are few orders of magnitude higher than that incirculation. Similar or identical FIN cDNA sequences have since beenidentified in plants, nematodes, rats, mice, and many other species,demonstrating that it is highly conserved along evolution (Guo et al.,2003).

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery that humanin and humaninanalogues protect pancreatic beta cells in vitro and in vivo fromapoptosis. Accordingly, humanin and its analogues are useful forpreventing and treating diabetes and promoting beta cell survival in anumber of applications.

In some embodiments, the invention is drawn to a method of treatingdiabetes in an individual in need thereof, comprising administering acomposition comprising humanin or a humanin analogue to said individualin an amount effective to improve survival of pancreatic beta cells,thereby treating diabetes. In some embodiments, the diabetes is type 1diabetes. In some embodiments, the diabetes is type 2 or gestationaldiabetes. In some embodiments, the composition is administered more thanonce, e.g., on a regular dosage schedule. Improved survival ofpancreatic beta cells is determined relative to a control, the design ofwhich is understood in the art. For example improved survival can bedetermined relative to survival of pancreatic beta cells in anindividual with diabetes, or an averaged value of survival in apopulation of individuals with diabetes.

In some embodiments, the invention is drawn to a method of preventingdiabetes in an individual in need thereof, comprising administering acomposition comprising humanin or a humanin analogue to said individualin an amount effective to improve survival of pancreatic beta cells,thereby preventing diabetes. In some embodiments, the individual haspre-diabetes or impaired glucose tolerance. In some embodiments, theindividual is at risk of developing diabetes. In some embodiments, thecomposition is administered more than once, e.g., on a regular dosageschedule.

In some embodiments, the invention is drawn to a method of improvingsurvival of a population of pancreatic beta cells, comprising contactingsaid population with a composition comprising humanin or a humaninanalogue in an amount effective to improve survival of said population,as compared to a control, e.g., an untreated population of pancreaticbeta cells. In some embodiments, the population of pancreatic beta cellscomprises cells from more than one individual. In some embodiments, thepopulation of pancreatic beta cells comprises cells from a singleindividual. In some embodiments, the cells are human. In someembodiments, the population of pancreatic beta cells is in an in vitroculture.

In some embodiments, the invention is drawn to improving survival of apancreatic beta cell transplant, comprising administering a compositioncomprising humanin or a humanin analogue to an individual with apancreatic beta cell transplant, in an amount effective to improvesurvival of said transplant as compared to a control. For example,survival can be compared to the survival of an untreated pancreatic celltransplant. In some embodiments, the composition is administered morethan once. In some embodiments, the composition is administered at thesame time as the transplant is performed. In some embodiments, thecomposition is administered after transplantation. In some embodiments,the transplant material is selected from the group consisting of a wholeorgan transplant (e.g., pancreas), a tissue graft, and a population ofcells, e.g., enriched in beta islet cells.

In some embodiments, the humanin analogue is selected from the groupconsisting of: S14G-HN; C8A-HN; D-Ser14-HN; AGA-HNG; AGA-(D-Ser14)-HN;AGA-(D-Ser14)-HN17; AGA-(C8R)-HNG17; EF-HN; EF-HNA; EF-HNG; EF-AGA-HNG;colivelin; P3R-HN; F6A-HN; F6A-HNG; F6AK21A-HNG; and Z-HN. In someembodiments, the humanin analogue is F6A-HNG (SEQ ID NO:16). In someembodiments, the composition comprises more than one humanin analogue,or humanin in combination with at least one humanin analogue.

In some embodiments, the composition is administered in combination witha second therapeutic, e.g., insulin. In some embodiments, thecomposition is administered at the same time as the second therapeuticcomposition. In some embodiments, the composition is administeredseparate from the second therapeutic composition, e.g., on anindependent dosing schedule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Humanin is a pancreatic beta cell survival factor. Serumstarvation induces apoptosis of NIT-1 cells. Increasing doses of humaninreduced the level of apoptosis. **=p<0.01 vs SF.

FIG. 2: Humanin activates ERK and STAT3. NIT-1 cells were incubated with100 nM HN in serum free media as indicated, cell lysates were harvested,and phosphorylated and total ERK1/2 and STAT3 were assessed byimmunoblotting.

FIG. 3: Humanin improves glucose tolerance in NOD mice. GlucoseTolerance Testing of humanin and saline-treated NOD mice (n=6 mice pergroup). **=p<0.01 by ANOVA.

FIG. 4: Humanin treatment decreased insulitis severity. Incidence andseverity of insulitis was analyzed busing formalin-fixed andparaffin-embedded pancreatic tissues. Sections were scored as describedin the Examples section.

FIG. 5: Humanin delays onset of diabetes in NOD mice. Percent survivalis indicative of a disease free state.

DETAILED DESCRIPTION OF THE INVENTION A. Introduction

The present invention involves a novel therapeutic method for treatingand preventing diabetes. Treatment with an effective amount of the smallpeptide humanin or one of several potent analogues of this peptide hasthe capacity of: a) preventing the onset of type 1 diabetes insusceptible individuals; b) sustaining the survival and activity ofinsulin-producing pancreatic beta cells; c) enhancing insulin action andglucose utilization in insulin responsive tissues such as fat. Humaninand humanin analogues act as suppressors of the development of type-1diabetes in the NOD mouse model and in vitro beta cell cultures, asdemonstrated herein. Humanin peptides and analogues have the potentialto serve as a single agent or as co-therapy with insulin or other agentsin various forms of diabetes or pre-diabetes.

The 24 amino acid humanin peptide was initially proposed to be aneuronal survival factor in the context of Alzheimer's disease. Humaninwas later shown to prevent cell death in certain non-neurological modelsas a Bax antagonist. The cellular receptor, if any, for humanin remainselusive, but may be related to FPRL-1. Humanin activates signalingcascades, including those involving Jak2 and STAT-3 (Matsuoka et al.(2006) CNS Drug Rev. 12:113-22). We found that humanin also bindsIGFBP-3. The interaction with IGFBP-3 is especially interesting asIGFBP-3 has been shown to induce insulin resistance both in vivo and invitro (Kim et al. (2007) Pediatric Res. 61:159-164). Furthermore, it waspreviously demonstrated that IGFBP-3 induces peripheral insulinresistance independent of IGF-1 binding.

Until we conducted our experiments, however, there was no suggestionthat humanin was related to diabetes.

The exact mechanism of humanin's protective activity and interactionwith IGFBP-3 may rely on: a) dimerization (Terashita et al. (2003) JNeurochem. 85:1521-38); b) FPRL-1 binding (Guo et al. (2003) Nature423:456-61); c) tyrosine kinase activation (Jung and Van Nostrand (2003)J Neurochem. 84:266-72), d) STAT-3 activation (Maximov et al. (2002) MedHypotheses 59:670-73); and e) antagonism of the pro-apoptotic moleculesBimEL and Bid (Caricasole et al. (2002) FASEB J. 16:1331-33; Tajima etal. (2002) Neurosci Lett. 324:227-31). In addition, TRIM11 plays a rolein the regulation of intracellular humanin levels throughubiquitin-mediated protein degradation pathways.

The invention is based on the discovery that humanin is a potent betacell survival factor in vitro and in vivo. Humanin is useful forpreventing diabetes in genetically at-risk individuals; treatingdiabetes, particularly type 1 diabetes; promoting islet transplantsurvival; stabilizing large donor sources of beta cells; and activatingendogenous beta cell regeneration with modulation of the autoimmuneresponse (a putative humanin receptor is found on white blood cells andmacrophages (Iribarren et al. (2005) Immunol Res., 31:165-76).

B. Definitions

As used herein, “diabetes” refers to the broad class of disorderscharacterized by impaired insulin production and glucose tolerance.Diabetes includes type 1 and type 2 diabetes (also called juvenile andadult-onset, respectively), gestational diabetes, prediabetes, insulinresistance, metabolic syndrome, and impaired glucose tolerance. Commonsymptoms include frequent urination, excessive thirst, extreme hunger,unusual weight loss, increased fatigue, irritability, and blurry vision.Diagnosis of these individual disorders is described in more detailbelow.

Type I diabetes is also known as Insulin Dependent Diabetes Mellitus(IDDM), Type 1 diabetes, and juvenile diabetes. The terms are usedinterchangeably herein. Treatment and diagnosis of the disease isdescribed in more detail below.

Humanin is a secreted peptide defined by the 24 amino acid sequence ofSEQ ID NO:1: MAPRGFSCLLLLTSEIDLPVKRRA. Humain also includessubstantially similar peptides and analogues, as defined herein. Humaninactivities include IGFBP-3 binding; inducing cell signaling and STAT-3activation; reducing apoptosis of neuronal cells; and improving survivalof pancreatic beta islet cells.

“Humanin analogues,” “humanin derivatives,” and equivalent terms, referto peptides with at least one humanin activity. Humanin analogues areoften more potent than humanin itself. One of skill can determinewhether any particular peptide is a humanin analogue by determiningwhether the peptide is capable of neuroprotection in an establishedhumanin assay, e.g., as described in Chiba et al. (2005) J. Neuroscience25:10252-61.

Generally, the humanin analogue comprises 17-50 amino acids comprisingthe amino acid sequence of SEQ ID NO:19. As used herein, an amino acidsequence providing the designation (x/y), as in SEQ ID NO:19, indicatesthat either amino acid x or amino acid y can be used at the indicatedposition. Analogues include, but are not limited to those shown in Table1.

TABLE 1 Humanin analogues NAME SEQ ID NO SEQUENCE Humanin 1MAPRGFSCLLLLTSEIDLPVKRRA S14G-HN (HNG) 2 MAPRGFSCLLLLTGEIDLPVKRRAC8A-HN (HNA) 3 MAPRGFSALLLLTSEIDLPVKRRA D-Ser14-HN 4MAPRGFSCLLLLTS*EIDLPVKRRA AGA-HNG 5 MAPAGASCLLLLTGEIDLPVKRRAAGA-(D-Ser14)-HN 6 MAPAGASCLLLLTS*EIDLPVKRRA AGA-(D-Ser14)-HN17 7PAGASCLLLLTS*EIDLP AGA-(C8R)-HNG17 8 PAGASRLLLLTGEIDLP EF-HN 9EFLIVIKSMAPRGFSCLLLLTSEIDLPVKRRA EF-HNA 10EFLIVIKSMAPRGFSALLLLTSEIDLPVKRRA EF-HNG 11EFLIVIKSMAPRGFSCLLLLTGEIDLPVKRRA EF-AGA-HNG 12EFLIVIKSMAPAGASCLLLLTGEIDLPVKRRA Colivelin 13SALLRSPIPA-PAGASRLLLLTGEIDLP P3R-HN 14 MARRGFSCLLLSTTATDLPVKRRT F6A-HN15 MAPRGASCLLLLTGEIDLPVKRRA F6A-HNG 16 MAPRGASCLLLLTGEIDLPVKRRAF6AK21A-HNG 17 MAPRGASCLLLLTGEIDLPVARRA Z-HN 18 MAKRGLNCLPHQVSEIDLSVQKRIConsensus sequence 19 (P/R/A)(R/A/G)(G/A)(F/A)S(C/R)LLL(L/S)T(S/T/G)(E/A)(I/T)DLP S* indicates D-Serine

Some of the humanin analogues have increased potency compared tohumanin, or slightly altered activities. Z-HN (SEQ ID NO:18) promotessurvival and activates STAT-3 and ERK in NIT cells with a two-foldgreater potency than humanin. F6AK21A-HNG (SEQ ID NO: 17) and F6A-HNG(SEQ ID NO:16) demonstrate similar activities with even greater potency.F6A-HNG, however, is devoid of IGFBP-3 binding activity.

“Pancreatic beta cells,” “beta islet cells,” and similar terms refer apopulation of pancreatic endocrine cells found in the Islets ofLangerhans. Beta islet cells produce and secrete insulin and amylin intothe bloodstream.

As used herein, “improving cell survival” refers to an increase in thenumber of cells that survive a given condition, as compared to acontrol, e.g., the number of cells that would survive the sameconditions in the absence of treatment. Conditions can be in vitro, invivo, ex vivo, or in situ. Improved cell survival can be expressed as acomparative value, e.g., twice as many cells survive if cell survival isimproved two-fold. Improved cell survival can result from a reduction inapoptosis, an increase in the life-span of the cell, or an improvementof cellular function and condition. In some embodiments, cell survivalis improved by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, ascompared to control levels. In some embodiments, cell survival is bytwo-, three-, four-, five-, or ten-fold of control levels.Alternatively, improved cell survival can be expressed as a percentagedecrease in apoptosis. In some embodiments, for example, apoptosis isreduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or up to 100%, as comparedto a control sample.

The term “preventing a disorder” as used herein, is not intended as anabsolute term. Instead, prevention, e.g., of type 1 diabetes, refers todelay of onset, reduced frequency of symptoms, or reduced severity ofsymptoms associated with the disorder. Prevention therefore refers to abroad range of prophylactic measures that will be understood by those inthe art. In some circumstances, the frequency and severity of symptomsis reduced to non-pathological levels, e.g., so that the individual doesnot need traditional insulin replacement therapy. In some circumstances,the symptoms of an individual receiving the compositions of theinvention are only 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% asfrequent or severe as symptoms experienced by an untreated individualwith the disorder.

Similarly, the term “treating a disorder” is not intended to be anabsolute term. In some aspects, the compositions of the invention seekto reduce the loss of insulin producing cells that lead to diabeticsymptoms. In some circumstances, treatment with the leads to an improvedprognosis or a reduction in the frequency or severity of symptoms.

“An individual in need of treatment or prevention” refers to anindividual that has been diagnosed with type 1 diabetes, type 2diabetes, gestational diabetes, pre-diabetes, insulin resistance,metabolic syndrome, or impaired glucose tolerance, or one that is atrisk of developing any of these disorders. Individuals in need oftreatment also include those that have suffered an injury, disease, orsurgical procedure affecting the pancreas, or individuals otherwiseimpaired in their ability to make insulin. Such individuals can be anymammal, e.g., human, dog, cat, horse, pig, sheep, bovine, mouse, rat,rabbit, or primate.

A “transplant,” as used herein, refers to the introduction of cells intoan individual (recipient or host). A pancreatic beta cell transplantrefers to a transplant that includes beta islet cells, but is notnecessarily composed entirely of beta islet cells. The transplantedcells can be introduced as an entire organ (e.g., a pancreas), a largelyintact tissue sample (e.g., a tissue graft), or as a disaggregatedpopulation of cells (e.g., enriched for beta islet cells) (Eisenbarth(2007) J. Clin. Endocrinol. & Metabol. 92:2403-07; King et al. (2007)Diabetes 56:2312-18). The introduced cells can be from anotherindividual (donor) or from the same individual. In some cases, cells areremoved from an individual, cultured under favorable conditions, andreplaced. In some cases, undifferentiated or partially differentiatedcells can be cultured under appropriate conditions to differentiate intobeta islet cells, and transplanted into an individual. See e.g., Yatohet al. (2007) Diabetes 56:1802-09; Jiang et al. (2007) Stem Cells25:1940-53; and Claibom and Stoffers (2008) Mt Sinai J Med 75:362-71.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that otherwise are expressed abnormally, under-expressed ornot expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state. It can be in either a dry or aqueoussolution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992);Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The terms “nucleicacid” and “polynucleotide” are used interchangeably.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins, wherein the amino acid residues are linked bycovalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but which functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)    -   (see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (e.g., a polyp eptide of the invention), which doesnot comprise additions or deletions, for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity overa specified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection, or across the entire sequence where not indicated. Theinvention provides polypeptides or polynucleotides encoding polypeptidesthat are substantially identical, or comprising sequences substantiallyidentical, to the polypeptides exemplified herein (e.g., humanin). Thisdefinition also refers to the complement of a nucleotide test sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

C. Expression and Purification of Polypeptides

Naturally-occurring, synthetic, or recombinant polypeptides of theinvention can be purified for use in compositions and functional assays.Naturally-occurring polypeptides of the invention can be purified fromany source. Recombinant polypeptides can be purified from any suitableexpression system (e.g., mammalian, insect, yeast, or bacterial cellculture).

The peptides of the present invention (i.e., humanin and humaninanalogues) may include both modified peptides and synthetic peptideanalogues. Peptides maybe modified to improve formulation and storageproperties, or to protect labile peptide bonds by incorporatingnon-peptidic structures. Peptides of the present invention may beprepared using methods known in the art. For example, peptides may beproduced by chemical synthesis, e.g., using solid phase techniquesand/or automated peptide synthesizers. In certain instances, peptidesmay be synthesized using solid phase strategies on an automated multiplepeptide synthesizer (Abimed AMS 422) using 9-fluorenylmethyloxycarbonyl(Fmoc) chemistry. The peptides can then be purified by reversedphase-HPLC and lyophilized.

For recombinant approaches, the present invention includes isolatednucleic acids encoding the polypeptides disclosed herein, expressionvectors comprising the nucleic acids, and host cells comprising theexpression vectors. More particularly, the invention provides isolatednucleic acids encoding humanin peptides and humanin peptide analogueshaving humanin activities, the peptides including, but not limited to,the peptides having a sequence selected from the group consisting of SEQID NOS:1-19.

When recombinant proteins are expressed by the transformed bacteria inlarge amounts, typically after promoter induction, although expressioncan be constitutive, the proteins may form insoluble aggregates. Thereare several protocols that are suitable for purification of proteininclusion bodies. For example, purification of aggregate proteins(hereinafter referred to as inclusion bodies) typically involves theextraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells typically, but not limited to, byincubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% NonidetP40, a non-ionic detergent. The cell suspension can be ground using aPolytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively,the cells can be sonicated on ice. Alternate methods of lysing bacteriaare described in Ausubel et al. and Sambrook et al., both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of the immunologically and/or biologically activeprotein of interest. After solubilization, the protein can be separatedfrom other bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify proteins from bacteriaperiplasm. Where the protein is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to those of skillin the art (see, Ausubel et al., supra). To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

The polypeptides of the invention may be purified to substantial purityby standard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., Current Protocols in Molecular Biology (1995supplement); and Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL,2nd Ed., (1989)).

A number of procedures can be employed when polypeptides are beingpurified. For example, polypeptides can be purified using ion exchangeor immunoaffinity columns.

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol is to add saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This will precipitate the most hydrophobic proteins. Theprecipitate is discarded (unless the protein of interest is hydrophobic)and ammonium sulfate is added to the supernatant to a concentrationknown to precipitate the protein of interest. The precipitate is thensolubilized in buffer and the excess salt removed if necessary, througheither dialysis or diafiltration. Other methods that rely on solubilityof proteins, such as cold ethanol precipitation, are well known to thoseof skill in the art and can be used to fractionate complex proteinmixtures.

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

The proteins of interest can also be separated from other proteins onthe basis of their size, net surface charge, hydrophobicity and affinityfor ligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art.

Immunoaffinity chromatography using antibodies raised to a variety ofaffinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc,hexahistidine, glutathione S transferase (GST) and the like can be usedto purify polypeptides. The His tag will also act as a chelating agentfor certain metals (e.g., Ni) and thus the metals can also be used topurify His-containing polypeptides. After purification, the tag isoptionally removed by specific proteolytic cleavage.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

D. Cell Culture and In Vitro Applications

In some embodiments, the compositions of the present invention are usedto improve the survival of pancreatic beta cells in culture. Cells to becultured include explants and primary and/or transformed cell culturesderived from patient tissues. Such methods are useful for maintainingand/or improving the viability of a donor source for transplant. In somecases, the population of pancreatic beta cells is expanded in culture.

Methods of cell culture are well known in the art. See, e.g., Freshneyet al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed.1994), and the references cited therein for a discussion of cell cultureconditions and how to isolate and culture cells from patients.Conditions for pancreatic cells in particular have been described(Lehmann et al. (2007) Diabetes 56:594-603 and King et al. (2007)Diabetes 56:2312-18).

In some embodiments, the cultured cells are initially undifferentiatedor partially differentiated. Conditions for differentiating cells intopancreatic beta cells are described in Yatoh et al. (2007) Diabetes56:1802-09 and Jiang et al. (2007) Stem Cells 25:1940-53.

This aspect of the present invention relies upon routine techniques inthe field of cell culture. In general, the cell culture environmentincludes consideration of such factors as the substrate for cell growth,cell density and cell contract, the gas phase, the medium, andtemperature.

Incubation of cells is generally performed under conditions known to beoptimal for cell survival. Such conditions may include, for example, atemperature of approximately 37° C. and a humidified atmospherecontaining approximately 5% CO₂. The duration of the incubation can varywidely, depending on the desired results. Proliferation is convenientlydetermined using ³H thymidine incorporation or BrdU labeling.

Plastic dishes, flasks, or roller bottles may be used to culture cellsaccording to the methods of the present invention. Suitable culturevessels include, for example, multi-well plates, Petri dishes, tissueculture tubes, flasks, roller bottles, and the like.

Cells are grown at optimal densities that are determined empiricallybased on the cell type. Cultured cells are normally grown in anincubator that provides a suitable temperature, e.g., the bodytemperature of the animal from which is the cells were obtained,accounting for regional variations in temperature. Generally, 37° C. isthe preferred temperature for cell culture. Most incubators arehumidified to approximately atmospheric conditions.

Defined cell media are available as packaged, premixed powders orpresterilized solutions. Examples of commonly used media include MEM-α,DME, RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium (see,e.g., GibcoBRL/Life Technologies Catalogue and Reference Guide; SigmaCatalogue). Typically, MEM-α or DMEM are used in the methods of theinvention. Defined cell culture media are often supplemented with 5-20%serum, typically heat inactivated serum. The culture medium is usuallybuffered to maintain the cells at a pH preferably from about 7.2 toabout 7.4. Other supplements to the media typically include, e.g.,antibiotics, amino acids, and sugars, and growth factors.

E. Pharmaceutical Compositions

The peptides of the present invention can be administered with asuitable pharmaceutical excipient as necessary. One of skill willunderstand that the composition will vary depending on mode ofadministration and dosage unit.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, diluents, tissue permeation enhancers,solubilizers, and the like. Preferably, the composition will containabout 0.01% to about 90%, about 0.1% to about 75%, about 0.1% to 50%, orabout 0.1% to 10% by weight of a conjugate of the present invention or acombination thereof, with the remainder consisting of suitablepharmaceutical carrier and/or excipients. Appropriate excipients can betailored to the particular composition and route of administration bymethods well known in the art. See, e.g., Remington's PharmaceuticalSciences, 18TH ED., Mack Publishing Co., Easton, Pa. (1990).

Examples of suitable excipients include, but are not limited to,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,saline, syrup, methylcellulose, ethylcellulose,hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols,e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The compositionscan additionally include lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying agents;suspending agents; preserving agents such as methyl-, ethyl-, andpropyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents suchas inorganic and organic acids and bases; sweetening agents; coloringagents; and flavoring agents. The compositions may also comprisebiodegradable polymer beads, dextran, and cyclodextrin inclusioncomplexes.

For oral administration, the compositions can be in the form of tablets,lozenges, capsules, emulsions, suspensions, solutions, syrups, sprays,powders, and sustained-release formulations. Suitable excipients fororal administration include pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, gelatin, sucrose, magnesium carbonate, and the like.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet, or capsule, and thus, the composition can contain, alongwith the conjugate or combination of conjugates, any of the following: adiluent such as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. Theconjugates can also be formulated into a suppository disposed, forexample, in a polyethylene glycol (PEG) carrier.

Liquid compositions can be prepared by dissolving or dispersing aconjugate or a combination of conjugates and optionally one or morepharmaceutically acceptable adjuvants in a carrier such as, for example,aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose,glycerol, ethanol, and the like, to form a solution or suspension, e.g.,for oral, topical, or intravenous administration. The conjugates of thepresent invention can also be formulated into a retention enema.

For topical administration, the compositions of the present inventioncan be in the form of emulsions, lotions, gels, creams, jellies,solutions, suspensions, ointments, and transdermal patches. For deliveryby inhalation, the composition can be delivered as a dry powder or inliquid form via a nebulizer. For parenteral administration, thecompositions can be in the form of sterile injectable solutions andsterile packaged powders. Preferably, injectable solutions areformulated at a pH of about 4.5 to about 7.5.

The compositions of the present invention can also be provided in alyophilized form. Such compositions may include a buffer, e.g.,bicarbonate, for reconstitution prior to administration, or the buffermay be included in the lyophilized composition for reconstitution with,e.g., water. The lyophilized composition may further comprise a suitablevasoconstrictor, e.g., epinephrine. The lyophilized composition can beprovided in a syringe, optionally packaged in combination with thebuffer for reconstitution, such that the reconstituted composition canbe immediately administered to a patient.

One of ordinary skill in the art understands that the dose administeredwill vary depending on a number of factors, including, but not limitedto, the particular peptide composition to be administered, the mode ofadministration, the type of application (e.g., prophylactic,therapeutic, etc.), the age of the patient, and the physical conditionof the patient. Preferably, the smallest dose and concentration requiredto produce the desired result should be used. Dosage can beappropriately adjusted for children, the elderly, debilitated patients,and patients with cardiac and/or liver disease. Further guidance can beobtained from studies known in the art using experimental animal modelsfor evaluating dosage. The humanin or humanin analog can be formulatedwithout undue experimentation for administration to a mammal, includinghumans, as appropriate for the particular application. Additionally,proper dosages of the compositions can be determined without undueexperimentation using standard dose-response protocols.

F. Methods of Administration

Administration of the peptides of the present invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,transdermal, intramuscular, oral, intra joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, or byinhalation. Administration can be targeted directly to pancreatictissue, e.g., via injection.

The compositions of the invention may be administered repeatedly, e.g.,at least 2, 3, 4, 5, 6, 7, 8, or more times, or the composition may beadministered by continuous infusion. Suitable sites of administrationinclude, but are not limited to, dermal, mucosal, bronchial,gastrointestinal, anal, vaginal, eye, and ear. The formulations may takethe form of solid, semi-solid, lyophilized powder, or liquid dosageforms, such as, for example, tablets, pills, lozenges, capsules,powders, solutions, suspensions, emulsions, suppositories, retentionenemas, creams, ointments, lotions, gels, aerosols, or the like,preferably in unit dosage forms suitable for simple administration ofprecise dosages.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects, each unit containing apredetermined quantity of active material calculated to produce thedesired onset, tolerability, and/or therapeutic effects, in associationwith a suitable pharmaceutical excipient (e.g., an ampoule). Inaddition, more concentrated compositions may be prepared, from which themore dilute unit dosage compositions may then be produced. The moreconcentrated compositions thus will contain substantially more than,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amountof a conjugate or a combination of conjugates.

Methods for preparing such dosage forms are known to those skilled inthe art (see, for example, Remington's Pharmaceutical Sciences, 18THED., Mack Publishing Co., Easton, Pa. (1990). The composition to beadministered contains a quantity of the peptides of the invention in apharmaceutically effective amount for improving beta islet cellsurvival. In addition, pharmaceutically acceptable salts of the peptidesof the present invention (e.g., acid addition salts) may be prepared andincluded in the compositions using standard procedures known to thoseskilled in the art of synthetic organic chemistry and described, e.g.,by March, Advanced Organic Chemistry: Reactions, Mechanisms andStructure, 4th Ed., New York, Wiley-Interscience (1992).

In another approach, nucleic acids encoding the polypeptides of theinvention are used for transfection of cells in vitro and in vivo. Thesenucleic acids can be inserted into any of a number of well-known vectorsfor the transfection of target cells and organisms as described below.The nucleic acids are transfected into cells, ex vivo or in vivo,through the interaction of the vector and the target cell. The nucleicacids, under the control of a promoter, then express a polypeptide ofthe present invention, thereby mitigating the effects of a diseaseassociated with reduced insulin production.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and other diseases in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science,256:808-813 (1992); Nabel et al., TIBTECH, 11:211-217 (1993); Mitani etal., TIBTECH, 11:162-166 (1993); Mulligan, Science, 926-932 (1993);Dillon, TIBTECH, 11:167-175 (1993); Miller, Nature, 357:455-460 (1992);Van Brunt, Biotechnology, 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience, 8:35-36 (1995); Kremer et al., BritishMedical Bulletin, 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy, 1:13-26 (1994)).

For delivery of nucleic acids, viral vectors may be used. Suitablevectors include, for example, herpes simplex virus vectors as describedin Lilley et al., Curr. Gene Ther., 1(4):339-58 (2001), alphavirus DNAand particle replicons as decribed in e.g., Polo et al., Dev. Biol.(Basel), 104:181-5 (2000), Epstein-Barr virus (EBV)-based plasmidvectors as described in, e.g., Mazda, Curr. Gene Ther., 2(3):379-92(2002), EBV replicon vector systems as described in e.g., Otomo et al.,J. Gene Med., 3(4):345-52 (2001), adeno-virus associated viruses fromrhesus monkeys as described in e.g., Gao et al., PNAS USA., 99(18):11854(2002), adenoviral and adeno-associated viral vectors as described ine.g., Nicklin et al., Curr. Gene Ther., 2(3):273-93 (2002). Othersuitable adeno-associated virus (AAV) vector systems can be readilyconstructed using techniques well known in the art (see, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; PCT Publication Nos. WO 92/01070 and WO93/03769; Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 (1988);Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, Current Opinion in Biotechnology 3:533-539 (1992); Muzyczka,Current Topics in Microbiol. and Immunol., 158:97-129 (1992); Kotin,Human Gene Therapy, 5:793-801 (1994); Shelling et al., Gene Therapy,1:165-169 (1994); and Zhou et al., J. Exp. Med., 179:1867-1875 (1994)).Additional suitable vectors include E1B gene-attenuated replicatingadenoviruses described in, e.g., Kim et al., Cancer Gene Ther.,9(9):725-36 (2002) and nonreplicating adenovirus vectors described ine.g., Pascual et al., J. Immunol., 160(9):4465-72 (1998) Exemplaryvectors can be constructed as disclosed by Okayama et al., Mol. Cell.Biol., 3:280 (1983).

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem., 268:6866-6869 (1993) andWagner et al., Proc. Natl. Acad. Sci. USA, 89:6099-6103 (1992), can alsobe used for gene delivery according to the methods of the invention.

In one illustrative embodiment, retroviruses provide a convenient andeffective platform for gene delivery systems. A selected nucleotidesequence encoding a polypeptide of the invention is inserted into avector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered to asubject. Suitable vectors include lentiviral vectors as described ine.g., Scherr et al., Curr. Gene Ther., 2(1):45-55 (2002). Additionalillustrative retroviral systems have been described (e.g., U.S. Pat. No.5,219,740; Miller et al., BioTechniques, 7:980-990 (1989); Miller, HumanGene Therapy, 1:5-14 (1990); Scarpa et al., Virology, 180:849-852(1991); Burns et al., Proc. Natl. Acad. Sci. USA, 90:8033-8037 (1993);and Boris-Lawrie et al., Curr. Opin. Genet. Develop., 3:102-109 (1993).

Other known viral-based delivery systems are described in, e.g.,Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA, 86:317-321 (1989);Flexner et al., Ann. N.Y. Acad. Sci., 569:86-103 (1989); Flexner et al.,Vaccine, 8:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques, 6:616-627 (1988);Rosenfeld et al., Science, 252:431-434 (1991); Kolls et al., Proc. Natl.Acad. Sci. USA, 91:215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad.Sci. USA, 90:11498-11502 (1993); Guzman et al., Circulation,88:2838-2848 (1993); Guzman et al., Cir. Res., 73:1202-1207 (1993); andLotze et al., Cancer Gene Ther., 9(8):692-9 (2002).

G. Therapeutic and Prophylactic Applications

In certain aspects, the compositions of the invention are used for thetreatment or prevention of a disease or disorder in a subject in needthereof. Examples of diseases or disorders suitable for treatment withthe humanin or humanin analogue compositions described herein include,but are not limited to, those disorders characterized by reduced bloodinsulin levels, or reduced number or function of pancreatic beta isletcells. Such disorders include type 1 and type 2 diabetes, gestationaldiabetes, pre-diabetes, insulin resistance, metabolic syndrome, andimpaired glucose tolerance. The compositions of the invention can beused prophylactically, e.g., for individuals with a geneticpredisposition for diabetes.

In some aspects, the compositions of the invention are used to improveprognosis for a beta cell transplant recipient. Humanin is capable ofimproving the survival of transplanted pancreatic beta cells, ascompared to untreated beta cell transplants.

Diabetes mellitus is associated with continuous and pathologicallyelevated blood glucose concentration; it is one of the leading causes ofdeath in the United States and is responsible for about 5% of allmortality. Diabetes is divided into two major sub-classes: Type 1 (alsoknown as Type I, juvenile diabetes, or Insulin-Dependent DiabetesMellitus (IDDM)) and Type 2 (also known as Type II, adult onsetdiabetes, or Non-Insulin-Dependent Diabetes Mellitus (NIDDM)).

The concentration of glucose in the human bloodstream must be controlledwithin a relatively tight range (60-120 milligrams per deciliter ofblood) to maintain normal health. If blood glucose drops too low, acondition known as hypoglycemia results, with symptoms such asfaintness, weakness, headache, confusion and personality changes.Excessive blood glucose, or hyperglycemia, may cause tissue damage dueto the chemical reactions between the excess glucose and proteins incells, tissues, and organs. This damage is thought to cause the diabeticcomplications of blindness, kidney failure, impotence, atherosclerosis,and increased vulnerability to infection.

Diabetes is usually diagnosed following the onset of excessive urinationor excessive thirst, often accompanied by weight loss. Often, patientswith newly-diagnosed type-1 diabetes have developed some degree ofdiabetic ketoacidosis by the time the diabetes is recognized. Secondarysymptoms include vision changes or unexplainable fatigue. Blood glucoselevel determination is necessary for an accurate diagnosis. Fastingblood glucose level determination is a standard approach used. However,the oral glucose tolerance test (OGTT) is considered to be moresensitive than fasted blood glucose level. Diabetes mellitus isassociated with impaired oral glucose tolerance (OGT). The OGTT thus canaid in the diagnosis (Emancipator (1997) Am J Clin Pathol 112:665 74;Type 2 Diabetes Mellitus, Decision Resources Inc., March 2000).

A “pre-diabetic individual” refers to an adult with a fasting bloodglucose level greater than 110 mg/dl but less than 126 mg/dl or a 2 hourPG reading of greater than 140 mg/dl but less than 200 mg/dl. A“diabetic individual” refers to an adult with a fasting blood glucoselevel greater than 126 mg/dl or a 2 hour PG reading of greater than 200mg/dl.

Impaired glucose tolerance is diagnosed in individuals that have fastingblood glucose levels less than those required for a diagnosis ofdiabetes, but have a plasma glucose response during the OGTT betweennormal and diabetics. Impaired glucose tolerance is considered aprediabetic condition, and impaired glucose tolerance (as defined by theOGTT) is a strong predictor for the development of Type II diabetesmellitus (Haffner (1997) Diabet Med 14 Suppl 3:S12 8).

The prediabetic state associated with glucose intolerance can also beassociated with a predisposition to abdominal obesity, insulinresistance, hyperlipidemia, and high blood pressure (Groop et al. (1997)Am J Hypertens 10(9 Pt 2):172S 180S; Haffner (1997) J DiabetesComplications 11:69 76; Beck-Nielsen et al. (1996) Diabet Med 13 (9Suppl 6):S78 84).

Type II diabetes mellitus is a progressive disease associated with thereduction of pancreatic function and/or other insulin-related processes,aggravated by increased plasma glucose levels. Thus, Type II diabetesmellitus usually has a prolonged prediabetic phase and variouspathophysiological mechanisms can lead to pathological hyperglycemia andimpaired glucose tolerance, for instance, abnormalities in glucoseutilization and effectiveness, insulin action and/or insulin productionin the prediabetic state (Goldberg (1998) Med Clin North Am 82:805 21).

Gestational diabetes mellitus (GDM) resembles type 2 diabetes in severalrespects, involving a combination of relatively inadequate insulinsecretion and responsiveness. It occurs in about 2%-5% of allpregnancies and may improve or disappear after delivery. Gestationaldiabetes is treatable but requires careful medical supervisionthroughout the pregnancy. About 20%-50% of affected women develop type 2diabetes later in life. While it may be transient, untreated gestationaldiabetes can damage the health of the fetus or mother. Risks to the babyinclude macrosomia (high birth weight), congenital cardiac and centralnervous system anomalies, and skeletal muscle malformations.

Early intervention in individuals at risk of developing diabetes,focusing on reducing the pathological hyperglycemia or impaired glucosetolerance may prevent or delay the progression towards diabetes andassociated complications. See, e.g., U.S. Pat. No. 7,109,174.

Insulin and sulfonylureas (oral hypoglycemia therapeutic agents) are thetwo major classes of diabetes medicines prescribed today in the UnitedStates. Insulin is prescribed for both Type 1 and Type 2 diabetes, whilesulfonylureas are usually prescribed for Type 2 diabetics only.Sulfonylureas stimulate natural insulin secretion and reduce insulinresistance; these compounds do not replace the function of insulin inmetabolism. Approximately one-third of patients who receive sulfonylureabecome resistant to it. Some Type II diabetics do not respond tosulonylurea therapy. Of patients who do respond to initial treatmentwith sulfonylureas, 5-10% are likely to experience a loss ofsulfonylurea effectiveness after about ten years. See, e.g., U.S. Pat.No. 7,115,284.

In addition, many anti-diabetic agents, for example, sulfonylureas andthiazolidinediones, have an undesired side effect of increasing bodyweight. Increased body weight in patients with prediabetic conditions orwith diagnosed Type II diabetes mellitus results in deleterious effectsdue to accentuation of the metabolic and endocrine dysregulation, andobesity per se is a pivotal risk factor for the development andprogressive worsening of Type II diabetes mellitus. Thus it is desirableto have an anti-diabetic agent which maintains or lowers body weight.See, e.g., U.S. Pat. No. 7,199,174.

One of skill in the art will appreciate that the humanin peptides andanalogues of the invention can be co-administered with other therapeuticagents for the treatment of diabetes. Co-administration can besimultaneous, e.g., in a single pharmaceutical composition or separatecompositions. The compositions of the invention can also administeredseparately from the other therapeutic agent(s), e.g., on an independentdosing schedule.

H. Transplantation of Pancreatic Beta Cells

Another approach for treatment of diabetes is transplant of pancreatictissue into an individual with reduced blood insulin levels. In somecases, an entire pancreas is transplanted, while in others, smallertissue grafts are used. Enriched populations of pancreatic beta isletcells can also be transplanted. Diabetic individuals often must receivemore than one transplant, as the insulin production of transplantedmaterial tends to decrease over time.

Cells for transplant are generally harvested from a donor individual orpopulation of individuals that are distinct from the recipient (orhost). Using these methods, immune suppression of the recipient isnecessary to prevent immune rejection by the recipient. Given theunwanted side effects of immunosuppression, however, interest is growingin culturing the recipient's own cells for reintroduction.

Cells to be transplanted can be treated with the compositions of theinvention before introduction into the host. Once the pancreatic betacells are transplanted, the compositions of the invention can beadministered to the host systemically or directly to the site oftransplantation, as described above.

Methods for culturing pancreatic beta cells are described above. Forreviews of transplant techniques, see, e.g., Claiborn and Stoffers(2008) Mt Sinai J Med 75:362-71; Eisenbarth (2007) J. Clin. Endocrinol.& Metabol. 92:2403-07; and references cited therein.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the invention is described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modificationsmay be made thereto without departing from the spirit or scope of theappended claims.

I. Examples 1. Dose-Dependent Protection of Beta Cells from SerumStarvation-Induced Apoptosis by Humanin

Secondary to the pro-survival effect of humanin observed in neuronalcells, we hypothesized that humanin could be a survival factor forneuroendocrine beta cells. Mouse NIT-1 insulinoma cells were serumstarved for 24 hours as control, and compared to cultures co-incubatedwith increasing doses of humanin ranging from 1 to 10000 nM.

Conditions were as follows: NIT-1 cells were purchased from ATCC andmaintained in F-12 Ham's medium (F12-K) supplemented with 10% FBS (fetalbovine serum), 10% L-glutamine, Penicillin (100 U/ml) and Streptomycin(100 μg/ml), in 25 cm² tissue culture flasks (Greiner, Frickenhausen,Germany). NIT-1 cells were maintained at 37 C in a 5% CO₂/95% airmixture and were passaged twice weekly by trypsination (Invitrogen lifetechnologies, Karlsruhe, Germany). For the experiment, NIT-1 cells wereseeded in 96-well plates at a density of 0.128×10⁵ cells per well. Afterabout 24 hours, medium was changed to FCS-free medium.

Apoptosis was quantified by a specific histone-associated DNA ELISA.Humanin potently protected beta cells from serum-starvation inducedapoptosis in a dose dependent manner (FIG. 1).

2. Humanin Inhibits Cytokine-Induced Beta Cell Apoptosis

Apoptosis is the main form of pancreatic beta cell death in animalmodels of type 1 diabetes mellitus. IFN-γ/TNF-α synergism has been shownto play an important role in autoimmune diabetes in vivo as well as betacell apoptosis in vitro. In this study, we used a caspase 3/7 specificfluorometric assay (ApoONE, Promega) to measure the degree of apoptosisinduction by cytokines in NIT-1 cells.

Cells were cultured as described above. For the experiment, NIT-1 cellswere seeded in 96-well plates at a density of 0.128×10⁵ cells per well.After about 24 hours, medium was changed to FCS-free medium and thecells were exposed to TNF-α (5 ng/mL) or were kept untreated ascontrols. As expected, TNF-α induced apoptosis in NIT-1 cells at 48hours.

Humanin protects beta cells from cytokine-induced apoptosis and serumstarvation induced apoptosis in a dose-dependent manner when measuredvia caspase 3/7 assay in doses that ranged from 10 to 1000 nM. TNF-αincreased apoptosis approximately 40% compared to serum-deprived cells.Humanin protected beta cells from TNF-α induced apoptosis in a dosedependent manner.

3. Humanin Activates ERK and STAT3 in Beta Cells

To investigate the mechanism of Humanin-induced beta cell survival, wetreated NIT-1 cells with 100 nM HN in serum free media. Cell lysateswere harvested and phosphorylated ERK1/2 and STAT3 were assessed byimmunoblotting.

Phosphorylation of ERK (activation) occurred 15 minutes after adding HN.Phosphorylation of STAT3 (activation) was a later event, 4 hours afteraddition of HN (FIG. 2).

4. In Vivo Treatment of NOD Mice with Humanin In Vivo Improves GlucoseTolerance

As proof of principle, we examined whether a 6-week course of daily IPinjected humanin (0.7 mg/kg/d) to euglycemic 9-week-old NOD mice(Taconic) could improve glucose tolerance as compared to saline-injectedcontrol mice (n=12/group). Daily IP injections were tolerated well.There was no difference in food intake or weight in humanin-treated vs.control mice. At the conclusion of the treatment, mice were fastedovernight and divided into groups that were subjected to IP insulin andglucose tolerance testing (n=6/group). Mice that were treated withhumanin showed a non-diabetic response to glucose challenge, whilecontrol mice showed the expected diabetic profile (FIG. 3). Response toIP insulin tolerance testing was similar in both groups, implying noeffect on peripheral insulin sensitivity. We additionally harvestedserum, pancreata, and various other organs from these mice forhistological studies and analysis of serum humanin, IGFBP-3, and otherIGF-related peptides.

5. Humanin Decreases Severity of Insulitis in the NOD Mouse

To test whether administration of humanin regulates the extent oflymphocyte infiltration into pancreatic islets, fixed and embeddedpancreatic tissues were examined. Pancreata from humanin-treated NODmice were formalin fixed, stained with hematoxylin/eosin, and assessedin a blinded fashion to determine the degree of lymphocyte infiltration.Results were compared to tissues from saline-injected control mice.Three or four randomly selected and nonadjacent sections from eachpancreas were scored independently by two blind observers and evaluatedfor mononuclear cell infiltration. Severity of infiltration wasclassified based on the degree of lymphocytic infiltration as follows:G0—normal (no infiltration); G1—peri-insulitis (mononuclear cellssurrounding islets and ducts but no infiltration of the isletarchitecture); G2—moderate insulitis (mononuclear cells infiltratingless than 50% of the islet architecture); and G3—severe insulitis (morethan 50% of the islet tissue infiltrated by lymphocytes, accompanied bya reduction in insulin staining). See Pastorale et al. (2002) Exp BiolMed (Maywood) 227:282-89.

Humanin treatment significantly decreased the total number of isletsinfiltrated by lymphocytes, as well as the severity of infiltration, ascompared to the vehicle-treated negative control mice (FIG. 4). Thus,humanin treatment partially blocks islet inflammation during type 1diabetes development in NOD mice. Our results indicate that humaninactivates endogenous beta cell regeneration and modulates the autoimmuneresponse. As noted above, a putative humanin receptor is found on whiteblood cells and macrophages.

6. Humanin Delays the Onset of Diabetes in the NOD Mouse

Four-week-old female NOD mice were treated with humanin. Beforetreatment, mice were tested two to three times per week for nonfastingblood glucose levels. Mice with blood glucose levels higher than 300mg/dL on three consecutive measurements were considered diabetic.Beginning at 5 weeks of age, two groups of NOD mice (n=25 each) wereadministered (0.7 mg/kg/d) synthetic humanin by IP injection daily(Bachem). NOD mice injected with the same volume of saline served asnegative controls. Over a 20-week treatment period, humaninsignificantly delayed the onset of diabetes (FIG. 5).

7. Humanin Analogue F6A-HNG Treats Type 2 Diabetes in NONcNZO10/LtJ Mice

F6A-HNG does not bind IGFBP-3. Mouse embryonic fibroblasts (MEFs)derived from normal (wild-type) and IGFBP3 deficient mice were used totest the effects of humanin and its analogues on survival. Apoptosis wasmeasured after 24 hours with 100 nM of each peptide by ELISA. F6A-HNGwas more potent in normal MEFs; however, HNG and F6A-HNG had equalpotency in IGFBP-3 deficient MEFs. The results demonstrate that F6A-HNGis more potent in this assay than humanin analogues that do bindIGFBP-3. The increased potency seems to result from the lack of IGFBP-3binding, because there is no additional advantage when IGFBP-3 is notpresent. Thus, F6A-HNG is distinct from previously described humaninderivatives.

NONcNZO10/LtJ mice are a model for obesity-induced type 2 diabetes.NONcNZO10/LtJ mice were injected IP with 25 μg F6A-HNG twice a day for14 days. Mice were subjected to 6 hour fast followed by an IP GTT onD15. Baseline glucose measurement was taken and the mice were injectedIP with 0.5 mg/g glucose. Glucose was sampled at 0 and 90, 120 min.

Mice treated with F6A-HNG displayed significantly better glucosetolerance. The glucose levels at 90 and 120 min were significantly lowerin the F6A-HNG treated mice. Thus, F6A-HNG and other humanin analoguesare useful in the treatment of type 2 diabetes, as well as the metabolicsyndrome associated with obesity.

What is claimed is:
 1. A method of treating type I diabetes in anindividual in need thereof, said method comprising: administering acomposition comprising a humanin analogue consisting of SEQ ID NO: 8 tosaid individual in an amount effective to improve survival of pancreaticbeta cells, as compared to an individual with type I diabetes, therebytreating type I diabetes.
 2. A method of improving survival of apopulation of pancreatic beta cells in vitro, said method comprising:contacting said population with a composition comprising a humaninanalogue consisting of SEQ ID NO: 8 in an amount effective to improvesurvival of said population, as compared to an untreated population ofpancreatic beta cells.